The present invention relates generally to detecting binding events and more particularly to a flow method for detecting binding events between a potential pharmaceutical chemical and a target binder using micro-x-ray fluorescence spectroscopy.
Pharmaceutical chemicals are the active ingredients in drugs such as the now popular Prilosec™, Lipitor™, Zocor™, Prozac™, Zoloft™, and Celebrex™, and it is believed that their pharmaceutical properties are linked to their ability to bind to the “binding site” of one or more proteins. The binding properties of a protein largely depend on the exposed surface amino acid residues of the polypeptide chain (see, for example, Bruce Alberts et al., “Molecular Biology of the Cell’, 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., “Molecular Cell Biology”, 4th edition, W. H. Freeman and Company, 2000). These amino acid residues can form weak noncovalent bonds with ions and other molecules. Effective binding generally requires the formation of many weak bonds at the “binding site” of the protein. The binding site is usually a cavity in the protein formed by a specific arrangement of amino acids. There must be a precise fit with the binding site for effective binding to occur. The shapes of binding sites may differ greatly among different proteins, and even among different conformations of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For these reasons, it is extremely difficult to predict which chemicals will bind effectively to proteins.
It can take many years to identify an effective pharmaceutical chemical. The desire to hasten the identification of important pharmaceutical chemicals is a constant challenge that has prompted the use or screening strategies for screening a large number of structurally or chemically related materials, known in the art as a “library”, for binding properties to proteins.
Screening methods generally involve combining potential pharmaceutical chemicals with target binders and determining which, if any, of the potential pharmaceutical chemicals bind to any of the target binders. Potential pharmaceutical chemicals are preferably water-soluble organic compounds that can dissolve into the blood stream. Target binders are generally biological materials such as enzymes, non-enzyme proteins, DNA, RNA, microorganisms (e.g. prions, viruses, bacteria, and the like), human cells, plant cells, animal cells, and the like. Potential pharmaceutical chemicals that bind to at least one target binder are likely candidates for further investigation of pharmaceutical properties (e.g. efficacy and toxicity).
Some of the known screening methods are described in the following three patents.
U.S. Pat. No. 6,147,344 to D. Allen Annis et al. entitled “Method for Identifying Compounds in a Chemical Mixture”, which issued Nov. 14, 2000, describe a method for automatically analyzing mass spectrographic data from mixtures of chemical compounds.
U.S. Pat. No. 6,344,334 to Jonathan A. Ellman et al. entitled “Pharmacophore Recombination for the Identification of Small Molecule Drug Lead Compounds”, which issued Feb. 5, 2002, describes a method for identifying a drug lead compound that inhibits binding of target biological molecules by contacting these target biological molecules with a library of cross-linked, target, binding fragments.
U.S. Pat. No. 6,395,169 to Ole Hindsgaul et al. entitled “Apparatus for Screening Compound Libraries”, which issued May 28, 2002, describes an apparatus that employs frontal chromatography combined with mass spectometry to identify and rank members of a library that bind to target receptor.
Screening methods sometimes employ tagged materials because the analogous untagged material is otherwise not visible using the analytical technique chosen for the screening method. Tagging may involve attaching a labeled chemical portion to a chemical. An example of a screening method requiring tags is fluorescence activated cell sorting. An example of this method involves preparing a solution of cells and antibodies bearing a fluorescent tag. Some of the antibodies bind to some of the cells. One at a time, the cells flow past a laser beam and a detector (such as a ultraviolet/visible fluorescence detector). Cells that fluoresce (are bound to the tagged antibodies) and are then deflected into a collector (see, for example, Bruce Alberts et al., “Molecular Biology of the Cell”, 2nd edition, Garland Publishing, Inc., New York, 1989, pages 159-160).
It is generally assumed that the attachment of a fluorescent tag only serves to make visible the otherwise invisible chemical and/or target binder, and does not alter the binding properties of the untagged analog. Since it is well known that even small changes to the structure of a chemical or target binder may affect its function, this assumption may not be a valid one. Tagged surrogates are structurally different from their untagged counterparts, and these structural differences could affect their binding properties.
An efficient method for screening potential pharmaceutical chemicals for binding to target binders remains highly desirable.